ABSTRACT This is a competitive renewal of R01EY024280, ?Developing Efficient AAV Vectors for Photoreceptor Targeting via the Vitreous?. Note that, due to advances made during the initial funding period, and our improved understanding of challenges in the field, our title has been changed to ?Engineering AAV for safe and efficient gene delivery to the human retina? to more broadly encompass our goals. FDA approval of an Adeno associated virus (AAV)- based gene therapy for RPE65-Leber congenital amaurosis (LCA2) solidified gene therapy?s place in current medical practice. However, injection of vector under the fovea of some patients led to central retinal thinning and loss of visual acuity. Similar decreases in retinal thickness were also observed in Choroideremia clinical trials. In more severe conditions, like X-linked Retinoschisis (XLRS), there is concern that subretinal injection (SRI) will further damage patient retinas. Since most inherited retinal diseases (IRDs) are caused by mutations in photoreceptor (PR)- and retinal pigment epithelial (RPE)- specific genes, development of gene therapies that more safely and efficiently target these cells remains a significant, unmet need. Targeting foveal cones is especially important, as they are responsible for acute, daylight vision. During the initial funding period, we developed AAV capsids capable of efficient retinal transduction following intravitreal injection (IVI) in primate. The inner limiting membrane (ILM) is the major barrier to AAV transduction via this this route. However, results from clinical trials utilizing IVI AAVs that show dose-limiting inflammation, and neutralization of the AAV capsid by pre-existing antibodies (NAbs) implicate the host immune system as a more immediate ?barrier? to clinical translation. The eye?s ?immune-privilege? has perhaps led to an under appreciation of the immune system?s role in shaping the outcome of intra-ocularly delivered AAVs. Naturally occurring antibodies to capsids capable of transducing retina via the vitreous (i.e. AAV2) are prevalent in up to 70% of humans. As such, a large percentage of patients will not meet inclusion criteria for emerging therapies. Here we propose experiments, based on strong preliminary data, to overcome these barriers. The majority of work will be performed in primates (macaque) as these barriers can only be recapitulated in intact eyes of animals with ocular characteristics and immune systems similar to humans. In Aim 1, we will enhance transduction and safety of intravitreally delivered AAVs by engineering the capsid and genome to avoid immune recognition. In Aim 2, we will enhance retinal transduction by subILM delivery of AAVs to enable efficient and specific transduction of inner and outer retina. In Aim 3, we will enhance transduction by subretinally delivered AAVs that spread laterally beyond the injection site. Vectors and methods investigated in this proposal will have an immediate impact on planned clinical trials to address inherited retinal diseases as well as non-orphan indications such as AMD. Development of these tools by academia (rather than industry) will ensure the availability of shared resources with the broader scientific community.